Synonymous mutations have traditionally been considered to be silent because they do not change the encoded amino acid. However, evidence is mounting that synonymous mutations can alter the structure, stability and/or function of mRNAs. Synonymous mutations have been implicated in cancer and Crohn?s disease, the development of antibiotic resistance, and bacterial adaptation to novel conditions. Few studies have investigated the mechanistic basis of the effects of synonymous mutations on mRNA structure, and investigations that have relied on computational predictions have often failed to identify the reasons for fitness effects. Further, the effects of non-synonymous mutations often ripple through the metabolic and regulatory networks in cells; there is no reason to think that synonymous mutations will affect only the encoded mRNA and nothing else. No previous study has addressed the system-wide effects of synonymous mutations. The goals of this project are 1) to identify high-impact synonymous mutations that increase the fitness of E. coli under strong selective pressures, and 2) to elucidate the mechanistic basis of these fitness effects at the system-wide and molecular levels. Aim 1 describes the introduction of all possible synonymous mutations into 300 E. coli genes encoding metabolic enzymes and transcriptional regulators ? a total of 312,000 mutations. The mutant cells will be screened under several different selective pressures to identify high-impact mutations. The vast scale of this screen sets this project apart from many previous investigations that have focused on mildly deleterious mutations in individual genes. Aim 2 addresses the effects of 30 high-impact synonymous mutations on the levels of the encoded mRNAs and proteins with the goal of identifying 10 that likely operate via different mechanisms. The system-wide effects of these 10 mutations will be investigated to help us understand why each mutation increases fitness under specific conditions. Aim 3 describes investigations of the molecular mechanisms by which 10 high-impact mutations affect the structure, stability and function of the encoded mRNAs. Possible mechanisms include creation of new transcriptional start sites, alteration of the binding of small regulatory RNAs, changes in structure that affect mRNA stability and/or translation, and changes in the structure of an encoded protein due to alterations of the tempo of translation, which can affect protein folding. This project will provide an unprecedented look at the frequencies and fitness effects of beneficial synonymous mutations and a detailed mechanistic understanding of the effects of 10 or more high-impact synonymous mutations that affect mRNA structure, stability and function in different ways.